一种HBV研究的新动物模型-喜马拉雅旱獭β-actin基因的克隆及序列分析  

Cloning of β-actin of M.Himalayana-A New Animal Model for HBV Study

作　　者：冯雪梅[1] 尹莹[1] 李安意[2] 朱珍妮[1] 陶元清[3] 王忠东[3] 陆蒙吉 杨东亮[1] 王宝菊[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院临床免疫研究室,武汉市430030 [2]华中科技大学同济医学院附属同济医院实验动物中心,武汉市430030 [3]青海省地方病预防控制所实验动物中心,西宁市811602 [4]德国Duisburg-Essen大学医学院病毒学研究所,德国埃森45122

出　　处：《医学分子生物学杂志》2011年第2期95-99,共5页Journal of Medical Molecular Biology

基　　金：国家重大传染病防治专项课题（No.2008ZX10002-011）

摘　　要：目的 喜马拉雅旱獭对土拨鼠肝炎病毒（woodchuck hepatitis virus,WHV）高度易感,可作为HBV感染的新动物模型.本研究对喜马拉雅旱獭β肌动蛋白（β-actin）的部分cDNA序列进行了克隆和序列分析,为喜马拉雅旱獭在HBV感染研究中的应用奠定基础.方法 根据Genbank的土拨鼠β-actin cDNA的序列设计特异性引物,提取旱獭脾组织总RNA作为模板,RT-PCR扩增旱獭β-actin cDNA序列;PCR产物纯化后连接至T载体（pMD18-T）,构建重组质粒pMD18-T-mhActin.对重组质粒进行PCR初筛及酶切鉴定,选择阳性克隆测序;对所获得的序列进行同源性和种系进化分析.结果 获得的旱獭β-actin序列为349 bp（nt887～1 235）,其中编码序列为323 bp（nt-887～1209）,编码106个氨基酸,包含形成二硫键的2个丝氨酸位点（氨基酸16和氨基酸105）及与β-actin功能相关的2个ATP结合位点、6个凝溶胶蛋白结合位点和6个profilin结合位点.同源性分析发现上述序列与其它哺乳动物β-actin的同源性均高达88 %以上,与土拨鼠β-actin的同源性最高（99.69 %）,其氨基酸序列的同源性为100 %.种系进化树分析提示喜马拉雅旱獭β-actin与土拨鼠β-actin的亲缘关系最近,其次为其它啮齿类动物.结论 成功克隆了喜马拉雅旱獭β-actin的部分序列.序列分析发现喜马拉雅旱獭β-actin与土拨鼠β-actin的同源性最高.Objective Marmata Himalayana （M. Himalayana）, which is highly susceptible to WHV infection, is a new animal model for HBV study. For further application of the animal model in HBV study, we cloned and analyzed the cDNA sequence of β-actin of M. Himalayana. Methods The primers were designed based on the cDNA sequence of woodchuck （M. monax） β-actin, which was published on Genebank. Total RNA was isolated from spleen tissues of M. Himalayana, and served as the template for RT-PCR amplification. PCR product was cloned into TA cloning vector with pMD18-T. Positive clones were selected and identified by PCR and restriction enzyme digestion. The recombinant plasmid pMD18-T-mhActin was determined by sequencing. The homology and phylogenetic tree of β-actin of many mammalian species including M. Himalayana and woodchuck were analyzed. Results Sequence analysis indicated that the 349bp sequence of mh-β-actin （re- ferred to nt887-1235 ） was cloned, including 323bp coding sequence （referred to nt887-1209 ） The protein sequence was predicted as 106aa, including 2 ATP binding sites, 6 Gelsolin binding sites and 6 profilin binding sites, which are closely related to the function of 18-actin. Homology and phylogenetic analysis indicated that β-actin of M. Himalayana has high homology （up to 88 % ） and close relationship to the counterpart of other mammalian species, with the highest homology （99.69 % ） and the closest relationship to the counterpart of woodchuck.Conclusion We successfully cloned β-actin of M. Himalayana. Sequence analysis indicated that β-actin of M. Himalayana has the highest homology and the closest relationship to the counterpart of woodchuck.

关 键 词：乙型肝炎病毒 喜马拉雅旱獭 肌动蛋白 序列分析 

分 类 号：R512.6[医药卫生—内科学]